Knockout moss

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Physcomitrella patens wild type (A) and knockout mosses produced therefrom (BD). Differing phenotypes in knockout mutants. Physcomitrella wild type and transformed plants were grown on minimal medium (English "knop medium") in orderto inducedifferentiation and gametophores . An overview (top row, size bar: 1 mm) and a close-up (bottom row, size bar: 0.5 mm) are shown for each plant. A, Haploid wild-type moss plant completely covered with gametophores and a close-up of a leaflet. BD, Various Mutants.

A knockout moss (engl. Knockout "knockdown") is a moss plant in which a targeted genetic alteration, the so-called gene targeting specifically one or more genes have been turned off ( gene knockout ). When a specific gene is lost, the knockout moss loses the property encoded by this gene. Due to the loss, one can now infer the function of the deactivated gene. This scientific approach is called reverse genetics , as the researcher wants to use a gene to explain its function. In classical genetics, however, the researcher starts from a phenotype and looks for the mutated gene. Knockout mosses are important in basic biological research and in biotechnology .

Scientific background

The targeted modification or the targeted switching off of genes is based on the integration of a short DNA strand at a precisely determinable position in the genome of the host cell. For this, the piece of DNA must have been constructed in such a way that it is identical to the gene locus at both ends . Then this DNA is integrated very efficiently via homologous recombination at this gene location. Knockout mice are also made according to this principle . So far, this method of targeted gene alteration, which in technical language gene targeting (is English gene targeting ) is, with plants only when the moss patens Physcomitrella established, since the efficiency of homologous recombination higher than in here several orders of magnitude seed plants is.

method

In order to specifically change genes in mosses, the DNA construct is incubated with protoplasts and with polyethylene glycol. Since mosses are haploid organisms , regenerating moss filaments ( protonemes ) can be checked directly for gene targeting, for example within just 6 weeks using PCR methods.

Examples

Plastid division

The first scientific publication to elucidate the function of a previously unknown gene using a knockout moss was presented in 1998 by Ralf Reski and colleagues. By knocking out the ftsZ gene, they succeeded in the first functional identification of a protein that is important for the division of organelles in eukaryotes .

Protein modifications

By knocking out several genes, Physcomitrella plants were created that lack the plant-specific glycosylation of proteins, an important post-transcriptional modification. These knockout mosses are used to manufacture complex biopharmaceuticals ( molecular pharming ) in moss bioreactors .

Mutant Collection

In cooperation with the company BASF , Ralf Reski and his colleagues have created a mutant collection of knockout mosses that are used for gene identification.

Individual evidence

  1. ↑ Egener et al. (2002): High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library. BMC Plant Biology 2, 6. doi : 10.1186 / 1471-2229-2-6
  2. ^ Ralf Reski (1998): Physcomitrella and Arabidopsis : the David and Goliath of reverse genetics. In: Trends in Plant Science. 3: 209-210. doi : 10.1016 / S1360-1385 (98) 01257-6
  3. ↑ A. Hohe, T. Egener, JM Lucht, H. Holtorf, C. Reinhard, G. Schween, R. Reski: An improved and highly standardized transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens. In: Current genetics. Volume 44, Number 6, January 2004, pp. 339-347, doi : 10.1007 / s00294-003-0458-4 , PMID 14586556 .
  4. ^ R. Strepp, S. Scholz, S. Kruse, V. Speth, R. Reski: Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. In: Proceedings of the National Academy of Sciences . Volume 95, Number 8, April 1998, pp. 4368-4373, PMID 9539743 , PMC 22495 (free full text).
  5. ↑ A. Koprivova, C. Stemmer, F. Altmann, A. Hoffmann, S. Kopriva, G. Gorr, R. Reski, EL Decker: Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. In: Plant biotechnology journal. Volume 2, Number 6, November 2004, pp. 517-523, doi : 10.1111 / j.1467-7652.2004.00100.x , PMID 17147624 .
  6. ↑ Tanja Egener, José Granado u. a .: High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library. In: BMC Plant Biology. 2, p. 6, doi : 10.1186 / 1471-2229-2-6 .
  7. ↑ Plant biotechnology: BASF and the University of Freiburg work together

literature

See also